Project Summary Influenza A virus is a highly contagious virus that causes upper and lower respiratory tract infections resulting in 200,000 hospitalizations and 36,000 deaths in the United States annually, and new influenza strains generate recurring epidemics and pandemics with significant attributable morbidity and mortality. Most of the mortality associated with influenza A virus infection is attributable to development of the acute respiratory distress syndrome (ARDS). Acute lung injury (ALI) and ARDS are defined by damage to the alveolar epithelium and endothelium, which allows the exudation of protein-rich fluid into the alveolar space. In our preliminary data, we show that vimentin, a type III intermediate filament protein, is required for the activation of the NLRP3 inflammasome. We provide preliminary data that vimentin?/? mice are protected from lung viral pneumonia following infection with influenza A virus (IAV). Increasing evidence from our group and others suggests that filamentous vimentin cytoskeletal structures play key roles in signal transduction pathways and provide a scaffold for the formation and activation of protein complexes, such as the NRLP3 inflammasome. We show that NLRP3 interacts with vimentin and that this protein-protein interaction is required for the processing and maturation of pro-IL-1? into biologically active IL-1?. Additionally, we provide preliminary data showing that vimentin is required for the interaction and translocation of NOD2 to the mitochondrial membrane, which results in the NOD2-mediated activation of IRF3 and release of interferon- from the IAV-infected cells. Based on these preliminary data, we hypothesize that vimentin acts as scaffold for the assembly and activation of the NLRP3 inflammasome and that NOD2 protein interaction with vimentin is required for the activation of IRF3 signaling. We have formulated three interrelated specific aims to study the regulation of vimentin intermediate filaments in both in vivo and in vitro models of influenza A virus-induced lung injury: Specific Aim 1: To determine the mechanism by which vimentin contributes to activation of NLR proteins during influenza A virus-induced acute lung injury. Specific Aim 2: To define the protein domain(s) in vimentin required for interaction with and activation of the NLRP3 inflammasome. Specific Aim 3: To determine whether the interaction between vimentin and NOD2 is required for the activation of IRF3 and the release of interferon from the IAV-infected cells.